mA-seq analysis of microRNAs reveals that the N6-methyladenosine modification of miR-21-5p affects its target expression.

In eukaryotes, N6-methyladenosine (mA) is one of the most abundant modifications on RNAs, and it plays important roles in many biological processes and diseases such as cancer. While most mA researches focus on message RNAs and long non-coding RNAs, recent studies have reported the presence of mA in small RNAs. Nevertheless, current knowledge about mA prevalence in mature microRNAs (miRNA) is extremely limited and the functional significance of mA methylation in miRNAs remains to be elucidated. Here, we demonstrated cell-specific mA profiles of miRNAs in A549 human non-small cell lung cancer (NSCLC) cells and HEK293A cells by using miRNA mA immunoprecipitation sequencing and constructed the consensus motif in mA-enriched miRNAs de novo. We found that miR-21-5p, an oncogenic miRNA, showed the highest mA enrichment in NSCLC cells. Depletion of the demethylase ALKBH5 did not change the expression level of miR-21-5p, but altered the mA abundance of miR-21-5p, thereby changing the expression levels of its target gene. We further synthesized mA modified miR-21-5p mimics in vitro and demonstrated that in NSCLC cells, mA marks in mature miR-21-5p could directly affect its silencing potency towards target genes, which finally impaired its promotion to proliferation and motility. Together, our findings reveal the landscape of mA modification in mature miRNAs, and provide the first evidence that it may contribute to the mRNA responses to cancer-related miRNAs.